AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 295: H21-H28, 2008. First published May 23, 2008; doi:10.1152/ajpheart.00135.2008
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TRANSLATIONAL PHYSIOLOGY

ATP/UTP activate cation-permeable channels with TRPC3/7 properties in rat cardiomyocytes

Julio Alvarez,1,2 Alain Coulombe,3 Olivier Cazorla,1 Mehmet Ugur,1 Jean-Michel Rauzier,1 Janos Magyar,4 Eve-Lyne Mathieu,5 Guylain Boulay,5 Rafael Souto,2 Patrice Bideaux,1 Guillermo Salazar,1 François Rassendren,6 Alain Lacampagne,1 Jérémy Fauconnier,1 and Guy Vassort1

1Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 637, Université Montpellier 1, Unité de Formation et de Recherche de Médecine, Centre Hospitalier Universitaire (CHU) Arnaud de Villeneuve, Montpellier, France; 2Laboratorio de Electrofisiología, Instituto de Cardiología, Havana, Cuba; 3INSERM, Unité Mixte de Recherche (UMR) S621, Université Pierre et Marie Curie, CHU Pitié-Salpêtrière, Paris, France; 4Department of Physiology, University of Debrecen, Debrecen, Hungary; 5Pharmacology, Faculty of Medicine, University of Sherbrooke, Fleurimont, Quebec, Canada; and 6Department of Pharmacology, Institut de Génomique Fonctionnelle, Centre National de la Recherche Scientifique UMR 5203, INSERM Unité 661, Université Montpellier I and Université Montpellier II, Montpellier Cedex, France

Submitted 7 February 2008 ; accepted in final form 16 May 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Extracellular purines and pyrimidines have major effects on cardiac rhythm and contraction. ATP/UTP are released during various physiopathological conditions, such as ischemia, and despite degradation by ectonucleotidases, their interstitial concentrations can markedly increase, a fact that is clearly associated with arrhythmia. In the present whole cell patch-clamp analysis on ventricular cardiomyocytes isolated from various mammalian species, ATP and UTP elicited a sustained, nonselective cationic current, IATP. UDP was ineffective, whereas 2'(3')-O-(4-benzoylbenzoyl)-ATP was active, suggesting that P2Y2 receptors are involved. IATP resulted from the binding of ATP4– to P2Y2 purinoceptors. IATP was maintained after ATP removal in the presence of guanosine 5'-[{gamma}-thio]triphosphate and was inhibited by U-73122, a PLC inhibitor. Single-channel openings are rather infrequent under basal conditions. ATP markedly increased opening probability, an effect prevented by U-73122. Two main conductance levels of 14 and 23 pS were easily distinguished. Similarly, in fura-2-loaded cardiomyocytes, Mn2+ quenching and Ba2+ influx were significant only in the presence of ATP or UTP. Adult rat ventricular cardiomyocytes expressed transient receptor potential channel TRPC1, -3, -4, and -7 mRNA and the TRPC3 and TRPC7 proteins that coimmunoprecipitated. Finally, the anti-TRPC3 antibody added to the patch pipette solution inhibited IATP. In conclusion, activation of P2Y2 receptors, via a G protein and stimulation of PLCβ, induces the opening of heteromeric TRPC3/7 channels, leading to a sustained, nonspecific cationic current. Such a depolarizing current could induce cell automaticity and trigger the arrhythmic events during an early infarct when ATP/UTP release occurs. These results emphasize a new, potentially deleterious role of TRPC channel activation.

purinergic receptor; signal transduction; infarction; arrhythmia

A HIGH-ENERGY PHOSPHATE DONOR, ATP has been extensively studied since the early description of a role for extracellular purines by Drury and Szent-Györgyi in 1929 (11). A low ATP concentration is present in the interstitial space despite its degradation by ectonucleotidases; moreover, its level can markedly increase during various physiopathological conditions (40). In particular, ATP and UTP are released during ischemia from various cell types, including cardiomyocytes (12), and this was shown to be associated with arrhythmia (21, 42). However, the mechanisms that lead to arrhythmia are unknown. This is of importance, since the early phase of arrhythmia during an ischemic period in patients is highly deleterious and is not sensitive to presently known pharmacological agents.

Extracellular ATP activates both the ionotropic (ligand gated) receptors of the P2X receptor family and the metabotropic (G protein coupled) receptors of the P2Y receptor family (40). The P2Y family is divided into two structurally distinct subfamilies. The first is composed of P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors, all coupled to Gq, which promotes PLC activation and diacylglycerol (DAG) production. The others, P2Y12, P2Y13, and P2Y14, are coupled to Gi, inhibiting adenylate cyclase (6, 14). Among the first subfamily, P2Y2, P2Y4, and P2Y6 also can be activated by UTP to various extents. P2 purinergic stimulation has multiple effects on cardiac ionic currents (40). On cells clamped at the resting potential, a fast application of ATP elicits a transient inward current that requires extracellular Mg2+ (8, 31, 32). Furthermore, during prolonged ATP application, in the presence or absence of Mg2+, after deactivation of the initial transient inward current, a weak sustained inward current can be recorded (31, 35). The nature of the channel protein that carries this later current is unknown.

Transient receptor potential channels (TRPCs) were first described in the phototransduction system of Drosophila melanogaster. Mammalian homologs encode channel proteins that have six transmembrane domains and assemble into heterotetramers (9, 28, 41). TRPCs are widely distributed in mammalian tissues and are involved in several cardiovascular functions and diseases (18, 25). Like P2X purinoceptors, most TRPCs are nonselective to cations and act to shift the membrane potential to around 0 mV, thus depolarizing cells from resting potential and allowing Ca2+ influx and cell automaticity. The TRPC subfamily is composed of seven members, TRPC1–7, with the TRPC3, TRPC6, and TRPC7 subgroup being directly activated by DAG (24). TRPC3- and TRPC7-expressing cells have been demonstrated to have both constitutively activated and ATP-enhanced inward currents that allow Ca2+ influx (17, 26). Recently, TRPC6 and TRPC6/7 have been identified as an essential part of the {alpha}1-adrenoceptor-activated cation currents in smooth muscle cells (19), whereas in heart, TRPC3 and TRPC6 proteins are essential for angiotensin II-induced hypertrophy (7, 27), and TRPC3 is necessary for the potentiated insulin-induced current (13).

In the present work at the cellular level, we have shown that ATP and UTP activate purinergic P2Y2 receptors. These receptors, via a G protein-dependent activation of PLCβ, trigger a fast-activating, sustained, nonselective cationic current through TRPC3/7 channels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental models and chemicals. Experiments were performed on cardiomyocytes isolated from adult male Wistar rats or as otherwise specified. Rats were killed by an intravenous injection of pentobarbital (100 mg/kg). The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996). The protocol was approved by the Animal Research Committee of INSERM. Cardiomyocytes were also isolated from postmyocardial infarcted (PMI) rats (1) and from control and transgenic mice deficient for the P2X1, P2X4, or both P2X1-P2X4 purinergic receptors (36) using a similar procedure, as well as from control dog and human according to Szabo et al. (39). All materials were purchased from Sigma-Aldrich except SKF-96365 (Calbiochem), U-73122 (Biomol), and fura-2 AM (Molecular Probes, Eugene, OR). Anti-TRPC (anti-TRPC3 and anti-TRPC6) antibodies raised against putative intracellular epitopes were obtained from Alomone Laboratories (Jerusalem, Israel), and the anti-TRPC7 antibody, a generous gift from Prof. W. P. Schilling (Case Western Reserve University School of Medicine, Cleveland, OH), was made against the sequence 843EKFGKNLNKDHLRVN857 and demonstrated to recognize no other TRPCs (15).

Electrophysiological experiments. Rat ventricular myocytes were enzymatically dissociated using a method previously described (4), kept in physiological solution containing (mM) 117 NaCl, 5.4 KCl, 1 CaCl2, 1.0 MgCl2, 10 glucose, and 10 HEPES, pH 7.4 at 22–24°C, and used within 6–8 h. Currents were recorded using the whole cell variant of the patch-clamp method at 22–24°C. K+ currents were blocked by Cs+. Compared with the physiological solution, the standard extracellular solution contained 20 CsCl, instead of KCl, and 2 CaCl2. The pipette (intracellular) solution contained (mM) 130 CsCl, 0.4 Na2GTP, 5 Na2ATP, 5 Na2-creatine phosphate, 11 EGTA, 4.7 CaCl2 (free Ca2+, 108 nM), and 10 HEPES, with pH adjusted to 7.2 with CsOH. For the experiments, cells were placed in petri dishes containing the same solution on the stage of an inverted microscope. A cell attached to the micropipette could be positioned on the extremity of each of six microcapillaries (250-µm inner diameter, Tygon microbore tubing; Norton Performance Plastics, Wayne, NJ) through which the different extracellular solutions were perfused by gravity at a rate of 0.1 ml/min. Solution changes were accomplished within 1 s. Alterations of the current at a holding potential of –80 mV were analyzed every 4 s. Currents were scaled to cell capacitance.

Single-channel recordings were performed with the classic cell-attached patch-clamp configuration. Only rod-shaped adherent cells isolated from rat heart with clear striations, sharp edges, without granulation, and showing no spontaneous contractile activity were chosen. Patch pipettes were pulled from borosilicate glass capillaries (Corning Kovar Sealing code 7052; WPI, Sarasota, FL) using a horizontal puller (DMZ-Universal Puller; Zeitz Instruments, Munich, Germany) and fire-polished before use. The pipette resistance was 5–10 M{Omega}. The currents were recorded using a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Foster City, CA) and filtered through an eight-pole Bessel low-pass filter 920LPF (Frequency Devices, Ottawa, IL) at a setting of 1 kHz (–3 dB point). Data were digitized at 5 kHz with a Digidata 1200 (Axon Instruments) using Acquis1 software (G. Sadoc, Centre National de la Recherche Scientifique, Gif/Yvette, France). Single-channel activity was recorded only when the seal resistance was ≥10 G{Omega}. All experiments were conducted at room temperature (20–24°C). Elementary conductances were determined as previously reported (10). Channel activity (mean patch current) was calculated by integrating current flow during the channel openings and dividing the integral by the total sampling time. The K+-rich medium in which cells were maintained before use contained (mM) 70 L-glutamic acid monopotassium salt, 25 KCl, 10 KH2PO4, 3 MgCl2, 0.5 EGTA, 10 HEPES, 20 glucose, and 10 taurine, and pH was adjusted to 7.4 with KOH. The superfusion solution contained (mM) 135 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 20 glucose, and pH was adjusted to 7.4 with NaOH. The pipette solution contained (mM) 135 NaCl, 4 KCl, 2 CaCl2, 10 HEPES, and 20 glucose, and pH was adjusted to 7.4 with NaOH. In some experiments, Na2ATP (100 µM) was added to the pipette solution.

Measurements of changes in intracellular Ca2+ concentration. Rat ventricular cardiomyocytes bathed in the physiological solution were loaded with fura-2 AM (2.5 µM) for 30 min at 35°C and then allowed to attach on a coverslip. Fluorescence images (2–4 rod-shaped cells in a field) were recorded and analyzed with a video image analysis system (MetaMorph 6.0/6.1; Universal Imaging). The fura-2 fluorescence image at an emission wavelength of 510 nm (bandwidth 20 nm) was followed at 22–24°C by excitation of fura-2 alternatively at 340 and 380 nm (bandwidth 11 nm) to obtain a 340/380 ratio on a pixel/pixel basis. Ca2+ was removed (Ca2+-free solution), and later Ba2+ was added (2 mM, Ba2+ solution).

Biochemical analyses. For RT-PCR, total RNA was extracted from myocytes or brain using TRIzol (Invitrogen) according to the manufacturer's specifications and reverse transcribed into cDNA using random hexamer (Amersham Pharmacia Biotech). An aliquot of the first-strand cDNA was used as a template for PCR, and rat TRPCs were amplified using the following primer sets: TRPC1, forward, 5'-CAAGATTTTGGGAAATTTCTAG-3', and reverse, 5'-TTTATCCTCATGATTTGC TAT-3'; TRPC3, forward, 5'-TGACTTCTGTTGTGCTCAAATATG-3', and reverse, 5'-CCTTCTGAAGTCTTCTCCTCCTGC-3'; TRPC4, forward, 5'-TCTGCAGATATCTCTGGGAAGAATGC-3', and reverse, 5'-AAGCTTTGTTCGAGCAAATTTCC-3'; TRPC5, forward, 5'-ATCTACTGCCTAGTACTACTGGCT-3', and reverse, 5'-CAGCATGGTCGGCAATGAGCTG-3'; TRPC6, forward, 5'-TCACTTGGAAGAACAGTGAAAGA-3', and reverse, 5'-CATCCTCAATTTCCTGGAATGAAC-3'; and TRPC7, forward, 5'-ACCTTCACAGACTACCCCAAAC-3', and reverse, 5'-GCCAAATATGGACCAAAACAAGG-3'. The resulting PCR product was analyzed using ethidium bromide-agarose gel electrophoresis and cloned into the pCR2.1-TOPO plasmid vector (Invitrogen) for sequencing analysis.

For Western blotting, adult rat cardiomyocytes isolated from left ventricles were quick-frozen and then homogenized. Proteins were heated for 5 min at 60°C in SDS sample buffer (2% SDS, 8 M urea, 0.08 M DTT, 0.05 M Tris, 1 mM EDTA, 1 mM EGTA, 0.5 mM PMSF, 10 µM E64, and 40 µM leupeptin). Proteins were separated by 7.5% SDS-PAGE, followed by transfer to 0.45-µm polyvinylidene diflouride membranes using standard techniques. The membranes were blocked with 3% bovine serum albumin (BSA) in 0.1% Tween 20-TBS (TBS-T). Membranes were labeled overnight with primary antibodies (anti-TRPC3 and anti-TRPC7, 1:200) in 0.1% BSA in TBS-T and washed with TBS-T before being labeled with 1:5,000 horseradish peroxidase-conjugated anti-rabbit antibody. Immunodetection was revealed with West Pico chemiluminescent substrate (Pierce Biotechnology). Quantification of signals was performed by densitometry using an imaging system (Kodak Image Station 2000R).

For the immunoprecipitation experiments, all reactions were performed during tumbling at 4°C. Cell protein extracts (1 mg) were precleared for 1 h with protein A-Sepharose CL-4B beads (Pharmacia) and then incubated for 1 h with 5 µl of antibody. Antibody-protein complexes were captured by the addition of protein A-Sepharose and incubated for 1 h to facilitate binding. Immunoprecipitated complexes were eluted from the beads using SDS sample buffer before SDS-PAGE and immunoblotting. Blots were visualized as described above.

Statistical analysis. Values are means ± SE of n cells. Statistical analysis was carried out using paired (comparing effects of agents on same cell) or unpaired (comparing effects between cells) Student's t-tests with the level of significance set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
P2Y2 purinergic receptor mediates the ATP/UTP effect in isolated cardiomyocytes. The mechanisms by which ATP triggers cardiac tissue automaticity, namely, the nature of the ATP-induced sustained inward current IATP and its signal transduction pathway, were investigated at the cellular level. The present study was mostly conducted in Mg2+-free extracellular solutions, taking advantage of the fact that IATP did not require the presence of Mg2+ for its activation. This allowed us to eliminate the initial transient surge of inward current (31). As shown in Fig. 1A, in the presence of Mg2+, application of 1 mM ATP triggered a fast downward change in the holding current that rapidly decreased. This transient inward current was followed by a sustained inward current. In the absence of Mg2+, only a sustained ATP-induced inward current, IATP, was seen. Furthermore, IATP was significantly increased in the Mg2+-free solution. IATP activated and deactivated within a minute on ATP application and withdrawal. IATP amplitude increased stepwise with cumulative ATP concentrations within the range from 30 µM to 3 mM (Fig. 1B). Under our experimental conditions that omitted Mg2+, IATP could be elicited by ATP and UTP with similar concentration dependency and amplitude range in adult ventricular cardiomyocytes isolated from control and transgenic mice deficient for the P2X1, P2X4, or both P2X1-P2X4 purinergic receptors and from PMI rats, as well as from control dog and human (Fig. 1C).


Figure 1
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  		Fig. 1. ATP and UTP activate a sustained inward current that is mediated by P2Y receptors. A: currents elicited at a holding potential (HP) of –80 mV on application of 1 mM ATP in the presence or absence of Mg2+ on a rat ventricular cardiomyocyte. The fast transient current required the presence of Mg2+ and was elicited only on fast ATP application. B: inward sustained currents (IATP) elicited at a HP of –80 mV by applying increasing concentrations of ATP. C: concentration-effect relationships of current density elicited by ATP or UTP in the absence of Mg2+ on ventricular cardiomyocytes isolated from control (n ≥ 9) and infarcted rats (PMI rat, n ≥ 6). The inset shows current densities induced by 2 ATP concentrations in control (n ≥ 4) and transgenic mice deficient for the P2X1, P2X4, or the P2X1-P2X4 purinoceptors (KO mice, n ≥ 3 for each single and double KO, pooled) and in dog (n ≥ 5) and human (n = 4) ventricular myocytes. Values are means ± SE; n is the number of cardiomyocytes from at least 2 hearts.

 
Under our experimental conditions, 2'(3')-O-(benzoylbenzoyl)ATP (BzATP) and adenosine-5'-[{gamma}-thio]triphosphate (ATP{gamma}S), both applied at 100 µM, were slightly less effective (70% of the ATP effect, n > 5 cells). Furthermore, whenever used at 30 µM or 1 mM, the ATP analogs adenylyl imidodiphosphate, adenosine 5'-(β,{gamma}-imido)triphosphate, and {alpha},β-methylene ATP (see also Fig. 6 in Ref. 32), as well as ADP, did not affect the holding current. UDP was also ineffective in triggering IATP. IATP exhibited a specific pharmacology that excluded it from being carried by any of the P2X receptors, including P2X7 (38). First, BzATP, described to be more potent than ATP to activate P2X7 receptor, induced a slightly weaker current. Second, the strong P2X inhibitors pyridoxal phosphate-6-azo(benzene-2',4'-disulfonic acid), oxidized ATP, and brilliant blue did not significantly affect IATP. However, suramin, a relatively selective P2Y1-P2Y2 antagonist, added at 100 µM, reduced by 45% the sustained current induced by 1 mM ATP (not shown; n > 4). Moreover, in cells patched with a pipette that contained a solution with 500 µM guanosine 5'-[{gamma}-thio]triphosphate (GTP{gamma}S), IATP occurred with similar kinetics and amplitude on ATP addition, whereas on ATP removal, the inward current was maintained in agreement with the metabotropic nature of the receptor (see Fig. 4B). Collectively, these data indicate that ATP and UTP elicited a sustained current through activation of the purinergic P2Y2 subtype receptors (40, 43).


Figure 4
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  		Fig. 4. Characteristics of the ATP-induced current. A: current-voltage relationship established after the application of 300 µM ATP in the absence of Mg2+ during a ramp potential as indicated at top. B: with 500 µM guanosine 5'-[{gamma}-thio]triphosphate (GTP{gamma}S) in the pipette solution, the brief application of ATP induced a current that was not reversible after ATP removal. Decreasing extracellular Ca2+ then significantly increased IATP amplitude. HP, –80 mV. C: mean sustained inward currents elicited under the conditions in B. Mean estimated chord conductances are 3.7 ± 0.8, 5.7 ± 1.2, and 7.3 ± 1.2 nS at 2, 0.6, and 0.1 mM extracellular Ca2+ concentrations, respectively (n = 5). D: the sustained ATP-induced current in Mg2+-free solution shared pharmacological properties with the expressed transient receptor potential channel TRPC7. Cyclopiazonic acid (CPA; 30 µM, incubated 10 min) and FK-506 (25 µM, incubated 10–30 min), both thought to increase intracellular Ca2+ concentration, inhibited the 300 µM ATP-induced current. La3+ (100 µM), Gd2+ (100 µM), and SKF-96375 (25 µM) induced a marked reversible inhibition of IATP, whereas flufenamic acid (FFA; 100 µM) significantly potentiated the sustained current. Values are presented as percentages of controls. The number of cells is indicated within bars for each experimental condition. *P < 0.05.

 
To elucidate the nature of the P2Y2 ligand and ATP2– or ATP4– (and UTP2– or UTP4–), we compared the effects of two external solutions with similar calculated free ATP4– and Ca2+ activities but varying ATP and MgCl2 concentrations. Except for the initial surge of transient current related to the presence of Mg2+, IATP had similar amplitude under these two experimental conditions, indicating that ATP4– was the P2Y2-specific agonist (Fig. 2A). At a constant 300 µM free external Ca2+ concentration ([Ca2+]o), the EC50 for the ATP4– effect determined in various ATP-containing but Mg2+-free solutions was 58 µM (Fig. 2B).


Figure 2
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  		Fig. 2. Free ATP4– is the agonist at the purinergic receptor. A: typical recording of the IATP in 2 solutions that contained both 2 mM Ca2+ and either 1 mM ATP and 0 mM Mg2+ or 3 mM ATP and 3.5 mM Mg2+, leading to similar estimated free ATP4– and Ca2+ activities (150 and 136 µM and 1.14 and 1.18 mM, respectively). Data are representative of 5 similar experiments. B: dose-response curve of IATP amplitude elicited by various ATP concentrations in a Mg2+-free, 300 µM Ca2+ solution (n = 6). The apparent half-effective ATP concentration (EC50ATP) was 558 µM, corresponding to a calculated EC50ATP4– of 58 µM. HP, –80 mV.

 
P2Y receptor activation triggers multiple signal transduction pathways in heart, including the production of DAG by various phospholipases (40). In the present experiments, activation of the sustained inward current by ATP was prevented by U-73122, a common PLC inhibitor, but not by its inactive analog, U-73433, or by propranolol and arachidonyltrifluoromethyl ketone, inhibitors of PLD and PLA2 cascades, respectively, to produce DAG (2). Furthermore, IATP was modified by neither LY-294,002 and wortmannin nor by genistein, respectively PLC{gamma}, phosphatidylinositol 3-kinase, and tyrosine kinase inhibitors (Fig. 3). Together, these data indicate that PLCβ was stimulated by the P2Y2 receptors through a G protein to produce DAG.


Figure 3
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  		Fig. 3. ATP-induced current requires the activation of PLCβ. Incubating the cardiomyocytes with the PLD inhibitor propranolol (200 µM), the PLA2 inhibitor arachidonyltrifluoromethyl ketone (AACOCF3; 50 µM, incubated 10–20 min), and the PLC{gamma} inhibitor LY-294,002 (100 µM, incubated 30 min), as well as with the broad-spectrum tyrosine kinase inhibitor genistein (20 µg/ml, incubated 30 min at 37°C), had no effect on the inward current elicited at –80 mV on the application of 300 µM ATP in the absence of Mg2+. IATP was prevented only in the presence of the PLCβ inhibitor U-73122 (10 µM, incubated 10–15 min). Filled bars represent experimental values, and open bars represent respective control cells; the number of cells is indicated above each bar. *P < 0.05.

 
Characteristics of the ATP/UTP-induced inward current in rat cardiomyocytes. The IATP-voltage relationship, recorded during a negative voltage ramp from +50 to –100 mV, exhibited a weak voltage dependence and showed a current reversal potential near 0 mV, indicating that the channel protein had a low selectivity for cations (Fig. 4A). Although a detailed analysis had not been performed, it was observed that the equimolar substitution of external Na+ by N-methyl-D-glucamine only slightly reduced (6 mV) the reversal potential of the ATP-induced current (n = 3).

To investigate the effect of various [Ca2+]o and avoid changing the active ATP4– concentration, we used a GTP{gamma}S-containing solution in the pipette and applied ATP for a short period that was, however, sufficient to activate the then maintained IATP. At that point, reducing Ca2+ in the perfusing solution markedly enhanced IATP. This effect was reversible and voltage independent with a twofold increase in chord conductance when [Ca2+]o was reduced 20-fold and without significant effect on the reversal potential (Fig. 4, B and C). This indicates that the channel was inhibited by external Ca2+ despite Ca2+ being allowed to pass through. Furthermore, cyclopiazonic acid (CPA) and FK-506, reported to increase [Ca2+]i, significantly reduced IATP (Fig. 4D). To further characterize the channel properties, we used various pharmacological compounds, namely, La3+, Gd3+, and the imidazole derivative SKF-96365, which were reported to inhibit Ca2+-permeable TRPCs, including the ATP-activated TRPC7 (3, 26). Likewise, these compounds significantly reduced the ATP-induced current in rat ventricular cardiomyocytes. Flufenamic acid, a common inhibitor of expressed homomeric TRPCs, except TRPC6 (19) (but see Ref. 5), slightly increased IATP (Fig. 4D). In addition, several compounds known to alter various currents in cardiac tissues, including lidocaine (100 µM), amiodarone (10–100 µM), quinidine (10–100 µM), and glibenclamide (50 µM), as well as isoproterenol (1 µM), did not significantly affect IATP (n ≥ 4).

Under control conditions in cell-attached patch clamp, the patch membrane continuously held at –80 mV demonstrated very rare single-channel openings. However, when the pipette contained 100 µM ATP, numerous openings were recorded (Fig. 5). The current reversed near 0 mV and showed no rectification. The two most frequently observed current levels exhibited conductances of 14 and 23 pS. In line with the above-reported signal transduction cascade, channel activity was markedly reduced when the cell was bathed in the presence of U-73122.


Figure 5
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  		Fig. 5. Single-channel characteristics of the ATP-induced current. A: no opening was observed in a patch from a control rat cardiomyocyte, whereas frequent openings were seen on another cell when the patch pipette contained 100 µM Na2ATP. Downward deflections of the current trace represent inwardly directed membrane currents at –80 mV. Note that bathing the cell in the presence of 10 µM U-73122 prevented channel openings. B: mean ATP-induced current in cell-attached patches on rat cardiomyocytes in control solution or in the presence of U-73122. The number of cells is indicated above each bar. C: mean single-channel current amplitudes as a function of membrane potential for the 2 most frequently observed low levels of current, determined from at least 14 membrane patches. The straight lines represent least squares fits of the data.

 
Further properties of the sustained cationic current were determined by microspectrofluorescence analysis. The application of 30 µM ATP, in the absence of Mg2+, was sufficient to trigger an influx of Mn2+, as indicated by a marked quenching of the fura-2 emissions following excitations at 340- and 380-nm wavelengths, whereas the signals varied in the opposite direction on ATP application in the control conditions as a consequence of Ca2+ influx and ATP-induced intracellular Ca2+ release (8, 30). Quenching was only very weak under basal conditions in the presence of Mn2+ before ATP application (Fig. 6A). We also used Ba2+ as a surrogate for Ca2+ to estimate cation influx. Adding Ba2+ to the Mg2+-free, Ca2+-free solution did not affect basal cell fluorescence. The further ATP application induced a significant Ba2+ influx (Fig. 6B). Ba2+ influx rate was similar in the presence of UTP, whereas UDP was inefficient to elicit Ba2+ influx. The ATP-induced Ba2+ influx was for the most part inhibited by U-73122 and reduced by one-half in the presence of 30 µM FK-506.


Figure 6
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  		Fig. 6. Fluorescence analysis of the ATP-induced current. A: original recordings in a rat ventricular cardiomyocyte loaded with fura-2. Changes in fluorescence induced by 30 µM ATP were in the opposite direction after excitation at either 340 or 380 nm, whereas there was a marked quenching effect after excitation at both wavelengths when the same ATP concentration was applied in the presence of 1 mM Mn2+ (3 similar experiments). A.U., arbitrary units. B: original tracings of the fluorescence ratio ({Delta}F340/380) in 3 rat ventricular cardiomyocytes sequentially submitted to Ca2+-free, 2 mM Ba2+-containing solution. A significant slow increase in Ba2+ fluorescence was observed only after 1 mM ATP application. C: pooled data of the maximal rate of increase in Ba2+ fluorescence induced by ATP, UTP, and UDP all at 1 mM or by 1 mM ATP on cells incubated with 10 µM U-73122 or 25 µM FK-506. The number of cells is indicated within bars for each experimental condition. *P < 0.05.

 
TRPC3/7 channel proteins carry the ATP/UTP-induced inward current. The presence of various TRPC mRNAs was checked in isolated adult rat ventricular cardiomyocytes. The mRNAs of TRPC1, TRPC3, TRPC4, and TRPC7, but not TRPC5 and TRPC6, were detected. Western blots further revealed the presence of TRPC3 and TRPC7 proteins having apparent molecular mass around 90–95 kDa. Furthermore, it was possible to immunoprecipitate TRPC7 with the anti-TRPC3 antibody, suggesting that both proteins contribute to heteromeric TRPC3/7 channels (Fig. 7). A band whose nature was not further checked was always seen around 170 kDa (n = 3). Anti-TRPC6 antibody did not reveal any protein.


Figure 7
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  		Fig. 7. Nature of the TRPC channel subunits. A: expression of TRPC mRNAs in cardiomyocytes isolated from adult rat compared with whole brain tissue indicates that TRPC1, TRPC3, TRPC4, and TRPC7, but not TRPC5 and TRPC6, were present in cardiomyocytes, whereas the efficacy of the anti-TRPC6 antibody had been controlled on brain tissue. RT, reverse transcription. B: Western blots reveal the TRPC3 and TRPC7 proteins in isolated rat cardiomyocytes. C: the TRPC3 antibody also immunoprecipitated (IP) TRPC7. The origin of the band at 171 kDa was not investigated.

 
The suggestion that TRPC3 contributes to the channel carrying the ATP-induced current was strengthened by the fact that adding the anti-TRPC3 antibody to the pipette solution markedly reduced IATP elicited in a rat ventricular cardiomyocyte under whole cell patch clamp. This inhibitory effect did not occur in the presence of the antigenic peptide and was not observed when the anti-TRPC6 antibody was used instead (Fig. 8).


Figure 8
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  		Fig. 8. Inhibition of the ATP-induced current by the anti-TRPC3 antibody. Pooled data represent the amplitude of the ATP-induced inward currents recorded within 5 min after the gigaseal formation in rat ventricular cardiomyocytes under whole cell patch clamp. The anti-TRPC3 antibody (Ab-C3) added to the pipette solution (1:200 dilution) significantly reduced IATP. Antibody-induced inhibition was prevented by further adding the TRPC3-antigenic peptide (Ab-C3+ Pep; 1:200) to the pipette solution. Adding the anti-TRPC6 antibody (Ab-C6) to the pipette solution did not significantly affect IATP. The number of cells is indicated within bars. *P < 0.05.

 

   DISCUSSION
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Extracellular purines and pyrimidines are released during various physiopathological conditions such as ischemia and are clearly associated with arrhythmia. In this article we report a series of events that could account for the triggering of arrhythmia by ATP-UTP. ATP (and UTP) in its free form binds P2Y2 purinoceptors, which via PLCβ activation trigger a sustained nonselective cationic current occurring through heteromeric TRPC3/7 channels.

TRPCs are assumed to be composed of four subunits that assemble to form homo- or heteromeric ion channels (16). The type of native channel formed depends on TRPC homologs endogenously expressed. The TRPC3/6/7 subfamily, besides having a high amino acid identity, is generally considered to be activated by a mechanism dependent on receptor-mediated activation of PLC and DAG production as initially reported for the activation of expressed TRPC3 and TRPC7 by ATP (17, 26). Recently, a similar cation channel with TRPC3/7 properties was shown to be activated by endothelin-1 in rabbit coronary artery myocytes (29). In the present work in ventricular cardiomyocytes, both TRPC3 and TRPC7 were expressed and coimmunoprecipitated. They both contributed to the ATP-induced current. Neither TRPC6 mRNA nor protein were observed at variance with TRPC6 protein occurrence in the mouse sinoatrial node (20) and in neonatal rat (27). In addition to the fact that our biochemical analyses were all performed on isolated rat cardiomyocytes, these different observations could be attributable to different tissues or species expression or to a shift in TRPC expression during development such as occurs in failing human heart (7). However, it is to be noted that the ATP/UTP-induced current had similar amplitude and dependency in PMI rat cardiomyocytes, as well as in P2X4-deficient mice. Indeed, these transgenic mice were investigated because the similarities of the current elicited by ATP in wild-type and P2X4-overexpressing transgenic mice led to the suggestion of a specific role of P2X4-mediated current in control mice (33). The contribution of TRPC3 to the cationic current-carrying channel was demonstrated by the fact that anti-TRPC3 antibody significantly reduced IATP. Such an anti-TRPC3 antibody applied on the internal membrane face was previously shown to produce a pronounced reduction of the TRPC3 properties in native constitutively active Ca2+-permeable channel in ear artery (3) and in 1-oleyl-2-acetyl glycerol (OAG)-treated mouse cardiomyocytes (13). Heterologously expressed TRPC7 was early reported to be activated by ATP (26). The contribution of TRPC7 to the ATP-evoked current in cardiomyocytes is strengthened by the observation that external Ca2+ ions have an inhibitory effect on the current amplitude as initially reported in heterologous expression systems, whereas it increases the TRPC6-carried current (34). In addition to the above-mentioned inhibition by external Ca2+, an increase in intracellular Ca2+ concentration ([Ca2+]i) after the application of CPA or FK-506 significantly reduced IATP as initially reported for expressed TRPC6 and TRPC7 (34) and heteromultimeric TRPC3-TRPC6 and TRPC6-TRPC7 channels in A7r5 smooth muscle cells (23). One cannot exclude the fact, however, that the elevated [Ca2+]i activates PKC or that FK-506 directly affects the TRPC channel behavior, since various immunophilins were reported to bind to TRPC proteins, more particularly FKBP-12 with TRPC3 and TRPC7 proteins (37).

In addition to being activated by a mechanism dependent on receptor-mediated activation of PLC and DAG production, TRPC3 and TRPC7, when overexpressed, often lead to increased basal Ca2+ level or increased Ba2+ and Mn2+ leak fluxes (17, 26, 45, 46) and even demonstrate activation by store depletion (22). In HEK-293 cells, TRPC1, TRPC3, and TRPC7 assemble to form native store-operated channels (SOCs), whereas TRC3 and TRPC7 can simultaneously participate in forming native store-operated and native DAG-stimulated channels (44). Recently, it also was suggested that TRPCs mediate store-operated Ca2+ channel activity to regulate mouse pacemaker firing (20). In isolated rat ventricular cardiomyocytes, heteromeric TRPC3/7 channels do not display constitutive or SOC activities, as indicated by very low single-channel openings in cell-attached patch and weak Mn2+ quenching and by a lack of Ba2+ influx in Ca2+-free medium.

In this work, ATP activated a current that was maintained in the presence of GTP{gamma}S. UTP was equally active, whereas UDP, an efficient agonist of the P2Y6 purinoceptor, was ineffective. These observations suggest the involvement of P2Y2 or P2Y4 receptor. The current was also activated by BzATP and ATP{gamma}S and showed much weaker inhibition by PPADS than by suramin. These findings further indicate that ATP/UTP binds to P2Y2 receptors on the basis of the pharmacological profiles, since after reexpression in oocytes, rat P2Y2, but not rat P2Y4, receptors are activated by BzATP and ATP{gamma}S and are more sensitive to suramin (43). Furthermore, activation of the P2Y2 receptors via a G protein leads to activation of PLCβ, as suggested by its inhibition by U-73122 but not by other PLC inhibitors. We also have clarified for the first time that only the free form of ATP, ATP4–, is the agonist at P2Y2 receptors by comparing the current amplitudes triggered by two solutions with similar calculated ATP4– and Ca2+ contents in the presence or absence of Mg2+, since to our knowledge Mg2+ ions have not been reported to alter TRPC3/7 channel conductance. This explains the increase in the sustained current amplitude observed on removing Mg2+ as shown in Fig. 1.

It is worth noting that the apparent EC50 of ATP to activate IATP via P2Y2 receptors was 58 µM, ~10–20 times the values determined on reexpressed rat P2Y2 receptor [2.7 and 3.6 µM for ATP and UTP, respectively (43)], and those for the ATP-induced modulations observed in Ca2+ and K+ currents whose purinoceptor subtypes were highly activated by 10 µM ATP are still unknown (40). This would allow physiological regulation before activation of this detrimental arrhythmogenic pathway. Our data suggest that in adult normal cardiomyocytes, IATP activates only in specific conditions, such as after a large surge of ATP/UTP release during infarction, at odds with the multiple physiological modulations of electrical and contractile activities induced by lower ATP and UTP concentrations (40, 42).

In conclusion, this work reveals a new, potentially deleterious role of TRPCs. Besides the multiple modulatory effects of ATP on electrical and contractile activities mediated by various pathways, we suggest that following the anomalous large release of ATP and UTP during early ischemic events, P2Y2 purinergic receptors stimulated by the free forms ATP4–/UTP4– activate heteromeric TRPC3/7 channels. The sustained inward current occurring through these channels induces cell depolarization and Ca2+ overload and possibly triggers arrhythmia. It is worth noting that, to some extent, other agonists that activate the DAG pathways and TRPCs could have some proarrhythmic activities; however, a specific arrhythmic effect of ATP should be considered as a consequence of its interstitial and potentially large release.


   GRANTS
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This work was supported, in most part, by the Institut National de la Santé et de la Recherche Médicale. J. Alvarez was supported by the French Embassy at Havana, Cuba. O. Cazorla and A. Lacampagne were supported by the Centre National de la Recherche Scientifique.


   ACKNOWLEDGMENTS
 
We thank D. S. Richard for stimulating support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Vassort, INSERM U-637, Physiopathologie cardiovasculaire, CHU Arnaud de Villeneuve, F-34295 Montpellier, France (e-mail: guy.vassort{at}inserm.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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